Altered abundances of human immunoglobulin M and immunoglobulin G subclasses in Alzheimer’s disease frontal cortex

The immune system has been described to play a role in the development of Alzheimer’s disease (AD), but the distribution of immunoglobulins and their subclasses in brain tissue has not been explored. In this study, examination of pathologically diagnosed frontal cortex gray matter revealed significantly higher levels of IgM and IgG in late-stage AD (Braak and Braak stages V and VI) compared to age-matched controls. While levels of IgG2 and IgG4 constant region fragments were higher in late-stage AD, concentration of native–state IgG4 with free Fc regions was increased in AD III and VI. RNA analysis did not support parenchymal B-cell production of IgG4 in AD III and V, indicating possible peripheral or meningeal B-cell involvement. Changes in the profile of IgM, IgG and IgG subclasses in AD frontal cortex may provide insight into understanding disease pathogenesis and progression.


IgG and IgM levels are different between late-stage AD and normal subjects.
To investigate the levels of IgM and IgG in different stages of AD brain tissue, supernatants produced from homogenized cortical gray matter were subjected to western blot analysis using anti-human antibodies for heavy chains Gamma (IgG-γ, Kruskal-Wallis test: p = 0.039) and Mu (IgM-µ, Kruskal-Wallis test: p = 0.006) (Fig. 1). The level of IgG-γ ( Fig. 1A) was significantly higher in AD V compared to Normal (p = 0.012). Similarly, the level of IgM-µ was significantly higher in late-stage AD (AD V, p = 0.002 and AD VI, p = 0.019) compared to Normal.

Figure 1.
Western blot analyses of human IgG gamma chain and human IgM mu chain were performed on denatured soluble proteins from cortical tissue supernatants of age-matched cognitively normal (Normal), AD III, AD V and AD VI subjects, with two samples duplicated on both membrane for normalization between membrane, denoted with filled rhombus and ☨, is presented as cropped band of interest (A). Compared to Normal, human IgG-γ levels were increased in AD V (B) and human IgM-µ levels were increased in both AD V and AD VI (C). Data was normalized using HRP-conjugated Beta-Actin monoclonal antibody and presented as mean ± SEM. Overall difference was determined with Kruskal-Wallis test and pairwise comparisons were conducted with Wilcoxon rank sum tests. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Concentrations of native-state IgM and IgG1-4 are altered in various stages of AD. Native-state
Ig isotypes in supernatants were quantified with Luminex Multi-Analyte Profiling technology. Using the Millipore Human Immunoglobulin Isotyping 5-plex assay, the presence and quantity of IgG1, IgG2, IgG3, IgG4, and IgM were measured (Kruskal-Wallis test : p = 0.436, p = 0.045, p = 0.366, p = 0.003, and p = 0.052, respectively) ( Fig. 4A,B). IgM achieved a borderline significance in the overall comparison and further pairwise comparisons showed that IgM protein levels demonstrated a significant increase in AD V (p = 0.026) and AD VI (p = 0.029) compared to Normal. IgM levels between AD III and Normal were not significantly altered. IgG2 levels were significantly elevated in AD VI (p = 0.019) compared to Normal. In the analysis of IgG4, 2 out of 5 AD III and 4 out of 5 AD VI cases from Duke had concentrations above the range of detection, and one Normal and one AD V sample from NSW were below the range of detection. Due to limitations in quantification, the maximum . Adjusted peptide abundance of IGHG2 (B) and IGHG4 (D) were higher in AD V and AD VI compared to Normal. Adjusted peptide abundance is presented as mean ± SEM. Overall difference was determined with Kruskal-Wallis test and pairwise comparisons were conducted with Wilcoxon rank sum tests. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. www.nature.com/scientificreports/ (458.14 ng/mL) and minimum (0.031 ng/mL) measurable concentrations were assigned to cases that were out of range, with the understanding that the actual values are much greater or lower than presented. Under these limitations, IgG4 levels in AD III (p = 0.033) and AD VI (p = 0.003) were significantly elevated compared to Normal. However, levels of IgG4 in AD V were on par with Normal.

Discussion
The relationship between the immune system and the progression of AD has recently been investigated 12,13 . However, the association between various Ig isotypes and subclasses in AD parenchyma has not been evaluated. By examining fresh-frozen frontal cortex (FCx) brain tissue from pathologically diagnosed AD (Braak and Braak stage III, V, and VI) and age-matched cognitively normal individuals (Normal), we demonstrated that the levels of Ig's were significantly altered in AD subjects compared to Normal. Furthermore, the trends of these Ig's with increasing Braak and Braak stages suggest a relationship with the pathological progression of AD. Immunoglobulin M (IgM) is the first antibody isotype produced by B-cells in an immune response that provides short term protection by opsonizing antigens/pathogens [30][31][32][33][34][35] . B-cells in the dura region of the meninges, either locally produced by the skull bone marrow or from the deep cervical lymph nodes, are genetically driven to produce IgM autoantibodies that assist in maintaining homeostasis 21,36 . Baulch and colleagues demonstrated a loss of B1 cells and their production of IgM-Aß, IgM autoantibodies targeting beta-amyloid (Aß) peptide 1-42, in the peripheral blood of 5XFAD mice compared to wild-type littermates 37 . Furthermore, the levels of IgM-Aß in peripheral blood of AD patients were also shown to be significantly lower than cognitively normal elderly individuals 15 . Our results demonstrate elevated levels of IgM in the FCx of late-stage AD (AD V and AD VI) compared to Normal, while IgM levels in early-stage AD (AD III) were comparable to Normal (Figs. 1B, 4B). Accumulation of proteins like Aß and phosphorylated Tau (p-Tau) in AD III is not as pronounced in the FCx as it is in the hippocampus, suggesting that at this pathological stage, an immunological response may not be warranted 5 . Increased protein accumulation in AD V FCx is accompanied with elevated IgM levels, which supports the idea that there may be a humoral immune response to AD pathology. This is compounded by the Table 1. RNA extracted from cognitively normal (Normal), AD III, AD V and AD VI cortical tissue samples were subjected to RT-qPCR to detect IGHG1, IGHG2, IGHG3 and IGHG4 RNA specific to each IgG isotype hinge region. After normalization using eukaryotic 18S rRNA, detection frequency is presented as fold changes using 2 −ΔΔCT method. www.nature.com/scientificreports/ consistently elevated levels of IgM that are detected in AD VI FCx. If IgM detected in the FCx are autoantibodies produced to maintain neuronal health, then the continually elevated levels exhibited in late-stage AD suggest a potential dysregulation in immunological response, resulting in the accumulation of protein and neuronal atrophy. Ig class switching is an important mechanism of an immune response that enables B-cells to switch isotype production from IgM to IgG, a higher affinity Ig that provides a tailored response against infection and long-term immunity 31,38 . Our results demonstrate elevated levels of IgG-γ in late-stage AD FCx, which supports the presence of an active and local humoral immune response in AD parenchyma (Fig. 1A), however, its relationship with disease severity remains undetermined. Kim and colleagues, using a 3xTgAD mouse model, demonstrated that an increase in activated B-cells in peripheral blood could infiltrate the parenchyma and increase pathology. In addition, the depletion of B-cells not only reduced the levels of Aß in the mouse brain tissue but also improved cognitive function, suggesting that cognitive impairment could be attributable to IgG and neuroinflammation 12 . Other researchers have demonstrated that removal of the humoral immune response in an AD transgenic mouse model accelerated AD pathology, which was ameliorated with administration of external IgG 13 . Furthermore, Figure 4. Native cortical tissue supernatants from age-matched cognitively Normal, AD III, AD V and AD VI subjects were quantified using the Human Immunoglobulin Isotyping Multiplex Assay for IgG1, IgG2, IgG3, IgG4 (A), and IgM (B). IgG2 concentrations were higher in AD VI compared to Normal, and IgG4 levels were increased in AD III and AD VI. IgM levels were higher in AD V and AD VI (B). Data is shown as mean ± SEM. Overall difference was determined with Kruskal-Wallis test and pairwise comparisons were conducted with Wilcoxon rank sum tests. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.  16 . These conflicting results may be dependent upon mouse models, IgG targets, or the IgG subclasses. Investigations of AD FCx were expanded to examine the distribution of the 4 IgG subclasses, and the roles they may play in the humoral response to AD pathology. Using various techniques, we demonstrated a change in IgG subclass profile in AD FCx compared to Normal, with significant increases in the levels of IgG2 and IgG4 (Figs. 2, 3, 4). IgG-Aß autoantibodies, typically found in the sera of AD patients and elderly individuals, are predominantly IgG1 and IgG3 subclasses, and have been suggested to have beneficial effects on amyloid pathology and neurotoxicity due to their pro-inflammatory effector functions 39,40 . Therefore, if IgG1 and IgG3 in AD parenchyma are autoantibodies, then their unaltered levels may indicate a possible failure in the humoral response due to skewed presence of other IgG subclasses.

Scientific
IgG2, the second most abundant IgG subclass in sera, was found to be elevated in late-stage AD parenchyma. While the relationship between IgG2 and neuronal disorders is unknown, it is suspected that the elevated levels may be related to increased IgG4 as suggested in other diseases [41][42][43] . IgG4 antibodies are generally formed in response to long-term antigen exposure in non-infectious situations and may become the dominant subclass 18 . IgG4 has a high affinity Fab region and has been shown to competitively bind to the target antigen to cause a "blocking" effect by preventing the binding of other effective Ig's 18,19,44,45 . Thus, in cases of chronic exposure to antigen, IgG4 may suppress an immunological response 45 . Significantly elevated IgG4 antibodies presented in AD FCx could act as autoantibodies by binding to proteins, such as Aß and p-Tau, thereby blocking other immunologically active Ig from binding to those antigens. This could inhibit the natural process of debris clearance and accelerate abnormal protein buildup, resulting in neurodegeneration. This, however, may not be the only pathway in which IgG4 may be deleterious.
Analysis of IgG4 with Luminex technology, which quantifies native-state Ig's by targeting the Fc region, demonstrated that the concentration of IgG4 in AD III subjects was significantly 4.8-fold higher compared to Normal (Fig. 4). This implies that IgG4 may play a role in the early stages of AD, prior to the toxic accumulation of Aß and p-Tau. Interestingly, the concentration of IgG4 in AD V subjects was similar to Normal, but increased by ninefold compared to Normal in AD VI. The drop in IgG4 concentration in AD V may be attributed to binding availability of the Fc region, since the levels were found to be consistently higher than normal when denaturing techniques were implemented. Additionally, the Fc region of IgG4 has been shown to bind to the Fc region of other Ig's, inhibiting their effector functions 18,19,[46][47][48][49] . This concept suggests that the Fc region of IgG4 in AD V FCx may be bound to a secondary Ig, obstructing an appropriate immunological response and consequently aiding the development of AD pathology.
It has been reported that a limited number of B-cells can reside in the parenchyma of healthy individuals, and their abundance increases with disease prevalence 36,50,51 . To explore whether local B-cells in the FCx were responsible for the observed Ig elevations, RNA was subjected to RT-qPCR. Detection frequency of IGHG2 and IGHG4 RNA transcripts in AD VI subjects were higher compared to Normal and earlier stages of disease (Table 1). This suggests that the IgG4 and IgG2 elevations exhibited in AD III and AD V may not be attributed to resident B-cells in the parenchyma but may be from meningeal B-cells or peripheral blood. In contrast, IGHG1 and IGHG3 RNA transcripts were detected among all groups evaluated. This suggests that local B-cells in AD parenchyma may be more inclined to produce IgG1 and IgG3 but not IgG2 and IgG4. The relationship between Table 2. Each immunoglobin isotype and subclass quantified from cortical tissue supernatants was compared between cognitively normal individuals (Normal) and AD individuals (AD, all stages combined) using Wilcoxon rank sum tests. Significantly altered levels are bolded. Data is represented as Mean, interquartile range [25th and 75th percentile].

Experiment
Full Sample (N = 25) Normal (N = 9) AD (N = 16) P value www.nature.com/scientificreports/ B-cells and AD needs further investigation to determine if isotype switching or IgG subclass production may be responsible for an improper immune response promoting neuroinflammation and disease pathogenesis. While our results demonstrated elevations in IgG2, IgG4, and IgM levels in AD brain tissue, we recognize limitations of our investigations. First, the sample size of each test group was limited, and the absence of AD IV samples precluded a sequential and more thorough analysis that would encompass the various stages of AD progression. Second, examining brain tissues from subjects with Mild Cognitive Impairment would have provided further insight into neuroinflammatory events and offered another perspective on the roles of Ig's and neurodegeneration in AD progression. Furthermore, in order to establish whether a distinct profile of Ig elevation is specific to AD, it would be beneficial to investigate other forms of neurodegenerative diseases to consider potential targets that are exclusive to AD.
This investigation presents the immune response as an upstream process to neuropathology in AD. Alteration in the Ig profile in AD parenchyma provides new insight into disease progression. Future investigations to clarify the targets of IgG4 and IgM may reveal their functions in AD pathogenesis, as well as opening new avenues in the study of neurodegeneration. Tissue processing. Brain homogenates were processed by sonicating (Polytron) 1 g of fresh-frozen cortical gray matter devoid of meninges and visible blood vessels in 20 mL of lysis buffer [tris buffered saline, pH 7.4 with 1 mM sodium orthovanadate, 10 mM sodium fluoride, 2 mM EGTA, and 0.1% Triton-X-100] containing complete mini protease inhibitor cocktail EDTA-free (Roche). Homogenates were centrifugated at 14,000 RPM for 10 min at 4 °C (Eppendorf 5418R centrifuge) to remove insoluble particulates. Supernatant fractions were used for analysis. Protein concentrations were measured using the Bradford assay (BioRad Laboratories) and Nanodrop (ND1000 Thermo Fisher Scientific).

Materials and methods
Western Blot analysis. Supernatants were examined under denaturing conditions; 20 µg of protein were incubated with dithiothreitol (DTT) and heated at 100 °C for 5 min. Samples were loaded on to 10% Bis-Tris mini protein gels (NuPAGE, Invitrogen) and electrophoretic separation of proteins was achieved with NuPAGE MOPS SDS running buffer (Invitrogen). Proteins were transferred to PVDF membranes (EMD Millipore) in the Mini Blot Module (Invitrogen) with NuPAGE transfer buffer (Invitrogen) containing 7.5% methanol over the span of one hour. Membranes were blocked with 5% nonfat dry milk in PBS with 0.01% Tween-20 for one hour at room temperature in order to minimize non-specific binding. Subsequently, membranes were incubated over night at 4 °C with either horseradish peroxidase (HRP) conjugated rabbit anti-human IgG heavy chain (IgG-γ) (Abcam, 1:5000), HRP conjugated rabbit anti-human IgM heavy chain (IgM-µ) (Millipore; 1:4000), rabbit anti-human IgG1 (Abcam, 1:5000), rabbit anti-human IgG2 (Abcam, 1:5000), rabbit anti-human IgG3 (Abcam, 1:5000), or rabbit anti-human IgG4 (Abcam, 1:5000) primary antibodies. It is important to note that the antibodies against human IgG1-IgG4 have a strong binding affinity and require independent experimentation. Membranes undergoing IgG subclass protein detection were washed and incubated with HRP-conjugated anti-rabbit secondary antibody for 2 h at room temperature (Invitrogen, 1:5000). A full list of antibodies and their specificity can be found in supplementary Table 2. Protein bands were visualized with chemiluminescent reagent and KwikQuant imager as per manufactuer instructions (Kindle Bioscience). Densitometric analysis was performed with ImageJ software. Normalization using a loading control antibody was achieved by incubating membranes with mouse anti-Actin monoclonal antibody (Millipore, 1:5000) followed by goat anti-mouse IgM (Millipore 1:2000), or HRP-conjugated mouse anti-Beta Actin monoclonal antibody (Invitrogen, 1:2000). The HRP conjugated anti-Beta Actin prominent upper band was quantified as per manufactuers' suggestion. To correct for technique variability, normalization was enabled by using two samples that were duplicated across the membranes, denoted with filled rhombus and ☨. Full length blots are provided in Supplementary Figs. 1 and 2. Proteomic analysis. Brain homogenates (100-300 µg) prepared as previously described were centrifugated at 13,000 RPM for 8 min at 4 °C (Sorvall Legend XTR centrifuge). The supernatants were collected, and reduced with 20 mM DTT at 56 °C for 30 min, followed by alkylation by subsequent addition of 40 mM iodoacetamide and a 20 min incubation in the dark. Trypsin digestion was performed using the S-trap digestion method. Briefly, 12% phosphoric acid was added at a 1.2% final concentration. Binding buffer (90% methanol/10 mM ammonium bicarbonate) was added in a 1:7 ratio and samples were loaded onto S-trap mini columns. These